Ion exchange process for separating proteins



3,073,747 IGN EXCHANGE PRQCESS F QR SEPARATING PRQTEHNS Ailen F. Reid, 5323 Harry Hines Blvd, Dalias 19, Tax. No Drawing. Fiied Dec. 13, 1958, er. No. 7231,2619 2 Claims. (Cl. 167-78) This invention relates to control of solubilities by ionexchange reactions and more particularly to varying salt concentrations in solutions by ion-exchange reactions to control the solubility of the solution. This case is a continuation-in-part of my co-pending application Serial No. 392,507 filed November 16, 1953, now abandoned, which was a continuation-impart of my application Serial No. 235,557 filed July 6, .1951 (now Patent 2,669,559), which in turn was a continuation-impart of my application Serial No. 20,583 filed April 12, 1948 (now abandoned),

It is an object of this invention to adjust the ion concentration in a solution of substances by ion-exchange re actions.

It is a further object of this invention to separate substances from a solution by varying the ion concentration in the solution.

It is more particularly the object of this invention to separate proteins whose solubilities differ from one 'another in solutions of various salt concentrations by adjusting the ion concentration of the salts in the solutions.

The process of the present invention consists broadly in an exchange of ions between a=solution and an ionexchange resin whereby selected ions are removed from the solution. This removal changing the concentration of the ions in the solution affects the solubilities of the other substances in the solution and the diiference in the solubilities is employed to cause a separation of the substances.

ion-exchange materials are solids which have the capacity of exchanging one ion for another in a solution contiguous to their surfaces. For example, if RH is the acid form of a cation-exchange resin in contact with a solution .of sodium chloride: y

and if R'OH is the hydroxyl form of an anion-exchange many ion-exchange materials are available in granular form suitable for packing in extended beds through which solutions may seep. To follow the application of such a column in adjusting ion concentration, consider the case of a long cation-exchange resin bed which has been equilibrated with a solution 0.1 M to sodium. For simplicity, the effect of hydrogen-ion concentration is neglected. In the top a solution 0.2 M to sodium is poured. The reaction (1) is displaced to the right with absorption by the resin of sodium ion and release to the solution of hydrogen ion in the attempt to restore equilibrium. The sodium concentration of the solution is reduced at the top of the column. This sodium depleted solution progresses further down the bed, its sodium concentration approaching nearer and nearer to 0.1 M until it exits at that concentration. More and more solution can be put through and have its concentration adjusted to 0.1 M until such time as the bed capacity for sodium under the operating conditions has become saturated far enough down so that the remaining layers of resin are not able to remove all of the excess sodium. A similar situation exists if the concentration of sodium in the solution introduced had been less than 0.1 M sodium from the resin going into the solution to make up the difference.

Similarly, an anion-exchange resin bed could adjust vwhich is soluble in distilled water.

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Patented Jan. 15, 1953 the concentration of any anions in a solution progressing through it. Thus with a mixture of the two types ofionexchangers, a solution can be'adjusted to concentrations of ions compatible with the capacity of the exchangers, if, prior to passage of that solution, the bed has been conditioned to be in equilibrium with the ion concentration desired.

Many natural proteins are soluble in aqueous solutions of common chemical compounds and are ofttimes found in mixtures in the same solutions from which one or several of them is desired to be removed. As an example, we may consider the fractions of human blood plasma. These include proteins known as ialbu mins which are soluble in distilled water, and various types of globulins which are not soluble in distilled water but are soluble in various concentrations of solutions of sodium chloride. When human blood serum is separated from the blood cells and fibrin, it contains these proteins in solutions which have a salt concentration of approximately 0.17 mole per-liter. The globulins present can be precipitated and the albumin left in the solutions if most of the salts are removed from this serum. Such fractionation is valuable because the separated albumin fractions can be used medicinally for the treatment of shock, etc. and the separated globulin contains several fractions which are medicinally useful as immune sera and in the treatment of hemophilic cases, etc. Because of the different solubilities of these components, it is evident that fractionation can be attained by adjusting the concentration of the non-protein constituents of the solutions. Up to the present time no practical method has been advanced for adjusting those concentrations for that purpose which was satisfactory from the standpoints vof expense and the protection of the chemically and thermally sensitive products- In order to adjust the concentration of these chemical constituents, I have introduced the solution at conservative temperatures to beds of ion-exchange resins which have been conditioned so that they will absorb ions. in the proper amounts and proportions to leave a concentration of ions in the solutions favorable for such fractionation. V

The introduction of such an improved method for this fractionation is important since present methods, .as Well as being quite expensive, involve processes such as the use of very cold alcohol which decreases the potency and desirability of some of the products.

The following is ,a procedure which may be followed in one application of the principle of this invention:

A mixture of a cation-exchange resin and an anionexchange resin is washed thoroughly with distilledwater to reduce the salt concentration. This mixture of resins is placed in a series of coltunns and washed again. The

solution of proteins to be separated, such as defibrinated ing the ion concentration of the solution changes the solubility of one or more of the proteins inthe solution with relation to the other proteins and consequently causes precipitation. The different proteins are precipitated at different points in the process astheir solubilities are different in the various ion concentrations.

Applying this .to human blood plasma, by removing salt from the plasma, the globulins are rendered insoluble 1 in .the solution andprecipitated, leaving the albumin The precipitated globulins may be centrifuged oiffrorn the'treated solution and those which remain trapped in the columns dissolved out with a saline solution. The final solution which has mi p 3' passed through all the beds contains no appreciable globulins after centrifugation. Albumin which is left in the precipitated globulin fractions may be dissolved out with distilled water without dissolving the globulins. The globulin fractions which have been washed out of the columns with the saline solution may contain some albumin because of the hold-up of liquid in the columns under normal operation.

The globulin fractions are separated from each other by a process of preferential leaching and precipitation. The mixture is first washed free of albumin and then leached with solutions of sodium chloride at a controlled pH. Under these conditions one globulin sub-fraction is dissolved preferentially from the others. The salt concentration and the pH of the leaching solution is then adjusted with an ion-exchange resin mixture and a selective precipitation of globulins is obtained. This globulin precipitate is subjected to another selective leaching followed by a selective precipitation. In this way, using proper control of the pH, the sodium chloride concentration and the concentration of other salts, it is possible to fractionate the globulins to any degree of refinement. In the described separation of globulin and albumin in blood plasma the mixture of two types of resins used is intended to keep the pH of the blood plasma solution from becoming either too alkaline or too acid. This is achieved by having the cation-exchange resin remove the sodium ion in this separation replacing it with hydrogen ion and the anion-exchange resin removing the chloride ion replacing it with a hydroxyl ion. The subsequent neutralization of the hydroxyl ions with the excess hydrogen ions and vice versa insures that no high concentration of acid is present in the solution. This is important as a highly acid pH may cause precipitation of the albumin and possibly denatu-ration of the proteins, and a basic pH would denature the proteins. The other blood serum salts are removed similarly. In the passage of the solutions through the ion-exchange resins some of the anion-exchange resin may be dissolved and taken into the solution. Pyrogens which are contaminants formed by bacteria may be present in the reconstituted plasma and could be present in the distilled water which is used in the operations. Any bacterial matter which might also be present could form other pyrogens unless the operations were carried out at an elevated temperature which would be objectionable as causing denaturation of the proteins. Therefore, to remove these impurities and contaminants from the solution it is passed through two final beds containing only the cation-exchange resin. These beds are conditioned first with distilled water and finally with pyrogen-free water and thus are able to take up the aforementioned contaminants. Finally, the solution is sent through a bacterial filter to elfectively remove any small particles of resin or bacteria which might have been picked up. This is removed by reprocessing. The albumin in the solution after concentration is safe for intravenous use.

The following example of plasma protein fractionation will serve as an illustration;

A mixture of equal parts of Amberlite IR 100, a cation exchange resin, and Amberlite IRA 400, an anion exchange resin, both furnished by the Rohm and Haas Company, Philadelphia, is washed thoroughly with distilled water until the salt concentration is less than four parts per million. The Amberlite IR 100 is in the hydrogen form and the Amberlite IRA 400 is in the hydroxyl form. Defibrinated blood plasma is mixed with this mixture. The salts are removed by the resins to a concentration of less than .001 ionic strength and the mixture of the resins maintains the pH between 7.4 and 5.5. Under these conditions most of the globulins precipitate out of the solution leaving essentially all of the albumins in solution.

A further application of the process of this invention is shown in removing hemoglobin from albumin. In samples of albumin a certain amount of hemoglobin which is released by the fracture of red blood cells is present and soluble in salt-free solutions. It is highly desirable to remove the hemoglobin, a highly colored impurity, from the albumin. The iron in hemoglobin may be bound to salicylate at values of pH between 7 and 5.5, conditions which will not precipitate albumin. By adjusting the concentrations of sodium salicylate and acid ions appropriately and then adding a mixture of cation and anion exchange resins which reduce the ionic strength of the solution while maintaining the pH between 7 and 5.5, the proteins from the hemoglobin contaminant are rendered insoluble and may be removed by centrifugation.

Another application of this invention is the separation of some of the proteins-in milk. Here a sample of con centrated milk whey containing lactoalbumin and lactoglobulin may be passed through a washed mixture of 70% Zeocarb H, an organic cation-exchange resin of the coal derivative type, furnished by the Permutit Company, and 30% Amberlite IR 4B, an amine type resinous anion exchanger containing approximately 14% nitrogen in the hydroxyl form, furnished by the Resinous Products Chemical Company. The mixture maintains the pH between 5 and 7. The salt-free solution contains primarily lactoalbumin, globulins being precipitated out as the salt concentration goes down.

Another example is in the separation of the globulins from the albumin in bovine serum. This is important as bovine albumin is routinely used in the medical sciences as a diagnostic aid. This separation may be accomplished by using either a mixture of four parts of Amberlite IR 100, a phenol-formaldehyde resin with a polyhydric phenol base and cationically active --SO H groups, and nine parts of Permutit Deacidite, a highly basic aliphatic amine type anion-exchange resin; or by using a mixture of one part of Amberlite IR and two parts of Amberlite IRA 400, a strongly basic anion-exchange resin with a high stability. When the concentration of the salts has been brought down to an ionic strength of .015 and has been maintained at a pH between 8 and 5 by treating the solution with a resin mixture, most of the globulins precipitate out leaving a solution which may be satisfactorily purified by a subsequent heat treatment.

Another application of the invention is the separation of proteins produced by the action of bacteria on culture media. These protein products may be used, when modifled somewhat chemically, as antigens to produce immunity in the body because of their stimulation of the production of antibodies. An example of this is dipththeria toxoid. In the production of this toxoid, Corynebacterium diphtheriae is allowed to grow in a culture medium. The toxicity of the resulting protein is destroyed by treatment with formaldehyde or some other modifying means while still preserving the antigenicity of the material. This detoxified protein must then be freed from other colloids present in order to make a preparation which is satisfactorily active and safe for injection. It is found that after the gross impurities have been separated from the toxoid, it may be further purified by subjecting its solution to a mixture of two parts Amberlite IRA 400 to one part Amberlite IR 100, thus reducing the salt concentration to below an ionic strength of .001 while keeping the pH value between 6.5 and 7.5. This treatment causes precipitation of contaminating colloid material which may then be mechanically separated from the rest of the proteins. The resulting protein may be even further separated by adjusting the pH with Amberlite IR 100 or a mixture of Amberlite IR 100 and a smaller proportion of Amberlite IRA/100 to a value of 3.5 where an electrophoretically pure compound is precipitated out. This may then be separated from the rest of the solution by mechanical separation.

A similar application of the invention exists in the purification of tetanus toxoid. After production in a similar manner by the action of Clostridium tetani on a nutrient medium and gross separation of the protein, the toxoid containing fraction may be purified from other protein rennin solution at a colloids by removal of the salts to an ionic strength of 0.001, keeping the pH value between 6.5 and 7.5 using a mixture of two parts of Amberlite IRA 400 to one part Amberlite IR 100. The precipitated impurities may be removed by mechanical separation. The remaining solution may be further separated by reducing the pH with Amberlite IR 100 or with a mixture of Amberlite IR 100 and a smaller proportion of Amberlite IRA 400 to 5.0 at which time an electrophoretically discrete protein component will precipitate out. This may be removed by mechanical separation.

Two important by-products of the meat packing industry are the enzymespepsin and rennin. They occur in the gastric secretions of such animals as cattle and hogs and are recovered from these sources. In the state in which they are recovered in the crude secretions they are not practical for commercial sale or usage. It is therefore necessary to purify them. A further application of this invention may be utilized in this purification. It is found that by using a mixture of about one part Amberlite IRA 400 to one part Amberlite IR 100 for the removal of salts and acid to an ionic strength of .001 and a pH of 3, many of the protein impurities are precipitated out from a pepsin solution. These may be removed by mechanical separation. On further treatment of the pepsin solution with Amberlite IRA 400 to bring the pH to 3.52, very concentrated pepsin will precipitate out. The mixture of Amberlites IRA 400 and IR 100 serves to keep the pH low enough so that the pepsin, which is unstable at a high pH, is not denatured.

As another modification of the procedure of the application of this invention, the reduction of the salt concentration of the solution of pepsin may be accomplished by first subjecting the solution to a small amount of Amberlite IR 100, not allowing the pH to go below 1, then transferring the solution to a small amount of Amberlite IRA 400, not allowing the pH to rise above 3, then transferring the solution to another batch of IR 100, then to another batch of IRA 400 and so on, always keeping the pH between the limits of 1 and 3 until the ionic strength has been reduced to 0.001. The solution is finally treated with Amberlite IRA 400 to bring the pH to 3.52. This treatment causes precipitation of concentrated pepsin as in the example above.

In a similar application of the invention, it a crude neutral pH is treated with a mixture of two parts Amberlite IRA 400 and one part Amberlite IR 100, the salt concentration may be brought to an ionic strength of .001 keeping the pH between 6.0 and 7.5. Many contaminating proteins are precipitated upon such treatment and may be removed by mechanical separation. Upon reduction of the pH to 3.75 by treatment with Amberlite IR 100 or a mixture of Amberlite IR 100 and Amberlite IRA 400, a concentrated rennin is precipitat-ed.

As another modification of the procedure of the ap plication of this invention, the reduction of the salt concentration of the solution of rennin may be accomplished by first subjecting the solution to a small amount of Amberlite IR 100, not allowing the pH to go below 5, then transferring the solution to a small amount of Amberlite IRA 4-00, not allowing the pH to rise above 8, then transferring the solution to another batch of IR 100, then to another batch of IR 400 and so on, always keeping the pH between the limits of 5 to 8 until the ionic strength has been reduced to .001. Finally the solution is brought to a pH of 3.75 by treatment with Amberlite IR 100 or a mixture of Amberlite IR 100 and Amberlite IRA 400. This treatment causes precipitation of contaminating colloid material as in the example above.

In the above described preparations, the enzymes retained their original activity. In other methods of removal or separation, such as the precipitation by saltingout with a high concentration of sodium sulphate, these enzymes may be denatured.

Separation of the proteins of peanuts, urease and wheat flour has been effective at pH values below 6. Many natural proteins are of vegetable origin and are quite important in the economic life of today, particularly in the food industry. For'example, it is found that the type of proteins present in ordinary wheat flour are very important in the use of this flour for the manufacture of products such as bread, cake, etc. Attempts to modify the concentrations of the proteins in flour so as to be able to market it for certain uses have been defeated by the failure to separate out some fractions from other protein constituents by a practical means. An application of this invention to this separation is so solubilize a portion of the wheat proteins in a salt solution and then to precipitate out certain proteins by removal of the salts using ion exchange resins. In a specific instance, some common wheat fiuor was mixed with a solution of salt with an ionic strength of approximately 0.15. "The resulting solution of wheat proteins was mechanically separated from the remaining solid material and subjected to a mixture of one part Amberlite IRA 400 and two parts of Amberlite IR 120, a strong cation exchange resin. As the salt concentration decreased with the pH also decreasing from 7 to 5, wheat proteins fractionally precipitated out. These could be mechanically separated from the solution.

Other natural vegetable proteins of considerable food value are found in peanuts. After the oil was removed from some crushed peanuts, some of the proteins were dissolved in a salt solution of an ionic strength of approximately .l5. Part of these proteinswere then fractionally precipitated by reducing the salt concentration to an ionic strength of less than .07 using a mixture of one part of Amberlite IRA 400 to one part of Amberlite IR with the pH being reduced from 7 to 4.5.

Certain vegetable proteins have enzymatic activity. One of the most important of these is urease which is made from jackbeans. After this protein has been separated grossly from the inactive material according to the most advanced means in common practice, the enzyme is still contaminated with much protein which serves not only to dilute its activity but also actually to'inhibit some of the enzyme action. If some of this grossly purified urease is placed in a solution of salt with an ionic strength of .15 and then subjected to a mixture of one part of Amberlite IRA 400 to one part of Amberlite IR 120 and the salt concentration brought to lower and lower ionic strength while the pH is brought down to between 6.5 and 4.5, some relatively inactive proteins will be first precipitated and later the urease will be precipitated in a much purer form.

In all of the instances cited, a modification of the process is to alternately treat the solution with the cation exchange resin not allowing the pH to go below the lower limit, and treat it with the anion exchange resin not allowing the H to go above the upper limit, continuing the alternation until the ionic strength and pH have reached the desired values.

I claim:

1. In a process of separating a protein from other proteins in an aqueous solution containing a plurality of proteins as solutes and also having a salt content present as cations and anions in a concentration rendering said first-mentioned protein soluble in said solution and in the absence of which cations and anions said protein is insoluble in said solution, another protein solute of said solution being soluble therein in the absence of said cations and anions in said concentration, the steps of providing a cation-exchange resin and an anion-exchange resin each conditioned to be in equilibrium with the anion concentration at which said solution-contained cations and anions are respectively removed from said solution being treated so that said resins will absorb from solution said corresponding ions, passing said aqueous solution of said proteins and salt solute alternately in contact with said cation-exchange resin and with said anion-exchange resin while holding the pH with each resin between limits effective to work the desired ion removal for the corresponding exchange and within the range pH 1 to pH 6 and thus removing from said solution said solubilizing cations by ion exchange with said cation-exchange resin to reduce said solubilizing cations by ion exchange with said cation-exchange resin and removing from said solution said solubilizing anions by ion exchange with said anion-exchange resin to reduce said solubilizing anions, and continuing said alternate exchanges to reduce the ionic strength of the solution thereby precipitating said first-mentioned protein from said solution of said proteins, and separating said precipitate from said solution.

2. A process of separating a protein from other proteins in an aqueous solution as set forth in claim 1 in which the aqueous solution is selected from the group consisting of diphtheria toxoid, tetanus toxoid, pepsin and rennin.

References Cited in the file of this patent UNITED STATES PATENTS 2,240,116 Holmes Apr. 29, 1941 2,461,505 Daniel Feb. 15, 1949 2,669,559 Reid Feb. 16, 1954 OTHER REFERENCES Anson: Advances in Protein Chemistry, vol. 3, pp. 414- UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No., 3,073,747 January 15, 1963 Allen F0 Reid 7 It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column 6 line 15 for ."so" read to 5 line 19, for

"fluor read flour same column 6, line 74, for "absorb" read sorb Signed and sealed this 27th day of August 1963 (SEAL) Attest:

DAVID L. LADD Commissioner of Patents ERNEST W. SWIDER Attesting Officer 

1. IN A PROCESS OF SEPARATING A PROTEIN FROM OTHER PROTEINS IN AN AQUEOUS SOLUTION CONTAINING A PLURALITY OF PROTEINS A SOLUTES AND ALSO HAVING A SALT CONTENT PRESENT AS CATIONS AND ANIONS IN A CONCENTRATION RENDERING SAID FIRST-MENTIONED PROTEIN SOLUBLE IN SAID SOLUTION AND IN THE ABSENCE OF WHICH CATIONS AND ANIONS SAID PROTEIN IS INSOLUBLE IN SAID SOLUTION, ANOTHER PROTEINS SOLUTE OF SAID SOLUTION BEING SOLUBLE THEREIN IN THE ABSENCE OF SAID CATIONS AND ANIONS IN SAID CONCENTRATION, THE STEPS OF PROVIDING A CATION-EXCHANGE RESIN AND AN ANION-EXCHANGE RESIN EACH CONDITIONED TO BE IN EQUILIBRIUM WITH THE ANION CONCENTRATION AT WHICH SAID SOLUTION-CONTAINED CATIONS AND ANIONS ARE RESPECTIVELY REMOVED FROM SAID SOLUTION BEING TREATED SO THAT SAID RESINS WILL ABSORB FROM SOLUTION SAID CORRESPONDING IONS, PASSING SAID AQUEOUS SOLUTION OF SAID PROTEINS AND SALT SOLUTE ALTERNATELY IN CONTACT WITH SAID CATIONS-EXCHANGE RESIN AND WITH SAID ANION-EXCHANGE RESIN WHILE HOLDING THE PH WITH EACH RESIN BETWEEN LIMITS EFFECTIVE TO WORK THE DESIRED ION REMOVAL FOR THE CORRESPONDING EXCHANGE AND WITHIN THE RANGE PH 1 TO PH 6 AND THUS REMOVING FROM SAID SOLUTIONS SAID SOLUBILIZING CATIONS BY ION EXCHANGE WITH SAID CATION-EXCHANGE RESIN TO REDUCE SAID SOLUBILIZING CATIONS BY ION EXCHANGE WITH SAID CATIONS-EXCHANGE RESIN AND REMOVING FROM SAID SOLUTION SAID SOLUBILIZING ANIONS BY ION EXCHANGE WITH SAID ANION-EXCHANGE RESIN TO REDUCE SAID SOLUBILIZING ANIONS, AND CONTINUING SAID ALTERNATE EXCHANGES TO REDUCE THE IONIC STRENGTH OF THE SOLUTION THEREBY PRECIPITATING SAID FIRST-MENTIONED PROTEIN FROM SAID SOLUTION OF SAID PROTEINS, AND SEPARATING SAID PRECIPITATE FROM SAID SOLUTION. 